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Mutagenesis studies toward understanding the intracellular signaling mechanism of antithrombin
Author(s) -
BAE J.S.,
REZAIE A. R.
Publication year - 2009
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2009.03337.x
Subject(s) - prostacyclin , intracellular , microbiology and biotechnology , antithrombin , chemistry , mutant , receptor , pertussis toxin , chemotaxis , heparin , pharmacology , biology , biochemistry , g protein , gene
Summary. Background: Recent studies have indicated that antithrombin (AT) possesses both anti‐inflammatory and antiangiogenic properties. Objectives: The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild‐type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin‐binding site. Methods: Direct cellular effects of the AT derivatives were compared in the LPS‐stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan‐4 or sphingosine 1‐phosphate receptor 1 (S1P 1 ). Furthermore, the roles of prostacyclin and nuclear factor (NF)‐κB in modulating these effects were investigated. Results: Both wild‐type and the RCL mutant, AT/Proth‐2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF‐κB pathway. Indomethacin abrogated both activities. The heparin‐binding site mutants, AT‐K114E and AT‐K125E, did not exhibit any protective activity in either one of these assays, but a potent pro‐apoptotic activity was observed for the AT‐K114E in endothelial cells. Both PTX and siRNA for syndecan‐4 inhibited the protective effect of AT, but the siRNA for S1P 1 was inconsequential. Conclusions: The interaction of AT with syndecan‐4 is required for its prostacyclin‐dependent protective effect through a PTX‐sensitive and non‐S1P 1 ‐related G i ‐protein coupled receptor. The RCL mutant, AT/Proth‐2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti‐inflammatory drug without increasing the risk of bleeding.